Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that catalyzes the transfer of triglycerides (TG) and cholesteryl esters (CE) between circulating lipoproteins (see Hesler, C. B., et. al. (1987) Purification and characterization of human plasma cholesteryl ester transfer protein. J. Biol. Chem. 262(5), 2275-2282), particularly between high density lipoproteins (HDL) and low density lipoproteins (LDL). The transfer of these neutral lipids is driven by their concentration gradients, hence net cholesteryl ester transfer occurs from HDL to very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and LDL with an obligate transfer of triglyceride in return. The mechanism by which these movements are facilitated is a matter of ongoing debate. The catalytic activities performed by CETP were identified as distinct from those performed by a related plasma protein, phospholipid transfer protein (PLTP), and subsequent research has demonstrated the independence of these two activities in vivo.
In humans, CETP plays a role in reverse cholesterol transport, the process whereby cholesterol is returned to the liver from peripheral tissues. Intriguingly, many animals do not possess CETP including animals with high HDL levels known to be resistant to coronary heart disease such as rodents (see Guyard-Dangremont, V., et. al., (1998) Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 120(3), 517-525). Numerous epidemiologic studies correlating the effects of natural variation in CETP activity with respect to coronary heart disease risk have been performed including studies on a small number of known human null mutations (see Hirano, K.-I., Yamashita, S. and Matsuzawa, Y. (2000) Pros and cons of inhibiting cholesteryl ester transfer protein, Curr. Opin. Lipidol. 11(6), 589-596). These studies have clearly demonstrated an inverse correlation between plasma HDL concentration and CETP activity (see Inazu, A., et. al. (2000) Cholesteryl ester transfer protein and atherosclerosis, Curr. Opin. Lipidol. 11(4), 389-396), leading to speculation that pharmacologic inhibition of CETP activity could be beneficial to humans by driving an improved circulating lipid profile.
However, epidemiologic studies have yet to provide an unequivocal validation of this concept. Importantly, the case of human CETP null mutants has provided evidence that the HDL formed in such humans is deficient in its interactions with the HDL receptor SR-B1 (see Ishigami, M., et al. (1994) Large and cholesteryl ester rich high density lipoproteins in cholesterol ester transfer protein deficiency can not prevent macrophages from cholesterol accumulation induced by acetylated low density lipoproteins, J. Biochem. (Tokyo) 116, 257-262), an effect that could undermine the benefit of associated HDL raising.
New classes of CETP inhibitors are being searched for using high throughput screening methods and are then being investigated with the goals of finding active inhibitors that may be useful as medications and that may validate inhibition of CETP as a method of reducing the risk of atherosclerosis by improving the HDL/LDL cholesterol ratio.
Assays that are currently in use for measuring the activity of CETP are not readily adapted to high throughput screening. The current invention provides a fluorescence assay that is sensitive enough to measure changes in the activity of CETP in the presence of compounds that are being screened as inhibitors. A fluorescence assay that was published by D. E. Epps et al. may be used, but the amount of “noise” in the measurements makes it very difficult to measure small changes in the transfer rate. See Epps, et al. (1995) Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein), Chem. Phys. Lipids, 77, 51-63. The current invention significantly improves the assay that was originally published by Epps, et al. The publication by Epps, et al. is incorporated by reference into this application.
In the method of Epps, a donor particle was made by incorporating a cholesteryl ester of a fluorescent BODIPY® dye (BODIPY-CE) into a synthetic HDL particle, along with human apoHDL, hen egg L-α-phosphatidylcholine, and hexabromotriolein (HBT). Acceptor particles were similar, except that they did not include BODIPY-CE, and they contained triolein rather than HBT. BODIPY is a fluorescent molecule which self-quenches in a concentration dependent manner. BODIPY-CE is a lipophilic non-polar cholesteryl ester which is similar enough to the cholesteryl esters in HDL and LDL to be transported by CETP. The rate of transfer of BODIPY-CE from donor particles to acceptor particles was determined by measuring changes in fluorescence as the BODIPY-CE migrated from the donor to the acceptor particle. BODIPY-CE fluoresces only minimally in the donor particle because of self-quenching. It becomes fluorescent as it migrates away from the donor particle to the LDL particle, where its concentration is low, so that it no longer self-quenches. However, there is typically enough background fluorescence from the BODIPY-CE in the donor particle that there is significant baseline noise, so that the measurements are very difficult to perform accurately.